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Biomedical subjects

E H Humphries

Publications and source records attributed to E H Humphries.

At least 37 records · Page 2Linked to original sources
PubMed1989

Selection for B cells with productive IgL gene rearrangements occurs in the bursa of Fabricius during chicken embryonic development.

The vast majority of immunoglobulin-expressing mature chicken B lymphocytes contain one functionally rearranged and one unrearranged allele of the immunoglobulin light chain (IgL) gene. Therefore, nearly all IgL V-J rearrangements present in mature chickens are in-frame. In contrast, the Ig genes of mature mammalian B cells contain a high proportion of out-of-frame V-J joints. To investigate the basis for this difference, gene rearrangement at the chicken IgL locus was characterized during embryonic development and in mature B-cell lines. Joining of the single functional variable (VL) segment with the single joining (JL) segment occurs in cells in multiple tissues during a transient period of chicken embryogenesis. Only one-third of the V-J joints cloned from days 10-12 of development are in-frame. An increasing proportion of in-frame V-J joints is observed within the bursa of Fabricius at successively later stages of development. Our data suggest that the bursa of Fabricius serves during embryonic development as a site of selective amplification of cells that have undergone productive V-J joining, such that nearly all V-J joints present in postembryonic B cells are in-frame. The high frequency of rearranged alleles joined in-frame that is found in posthatching bursal cells and mature B-cell lines appears to result from a low frequency with which cells undergo IgL rearrangement at both alleles, rather than from an increase in the precision of V-J joining in avian species.

Alleles↗
PubMed1989

RAV-1 insertional mutagenesis: disruption of the c-myb locus and development of avian B-cell lymphomas.

Infection of young chickens with RAV-1, a subgroup A isolate of avian leukosis virus, results in the development of lymphoid leukosis, a B-cell lymphoma characterized by provirus insertion into the c-myc locus. We report here that when 12- to 13-day-old embryos rather than 1-day-old chickens were infected with RAV-1, a novel B-cell lymphoma developed in which proviral insertions had activated expression of the c-myb gene. These tumors expressed elevated levels of a 4.5-kilobase myb-containing mRNA transcript that contained c-myb sequences not found in v-myb. The c-myc locus in these tumors appeared normal. The biological properties of the activated myb lymphoma were distinct from those of lymphoid leukosis. Metastatic disease developed within 7 weeks of infection. Distinct intermediate pathogenic stages with preneoplastic and primary neoplastic lesions were not detected. Although bursal tissues appeared to be nonmalignant on gross examination, Southern analyses of bursal DNA revealed the presence of tumor with the same clonal origin as abdominal lymphoma masses. The dependence on embryonic infection for development of activated myb lymphoma suggests that the target cells in which c-myb is activated are found only in embryos and are distinct from those cells that give rise to lymphoid leukosis.

Animals↗
PubMed1988

A nonimmunosuppressive helper virus allows high efficiency induction of B cell lymphomas by reticuloendotheliosis virus strain T.

We have documented the effect of two nondefective helper viruses, reticuloendotheliosis virus A (REV-A) and chick syncytial virus (CSV) infection on bursal tissue. REV-A infection results in bursal atrophy, destroying both its structural and functional integrity. In contrast, the bursae in CSV-infected chicks, while reduced slightly in size, appear both structurally and functionally normal. REV-A-induced bursal atrophy is not a result of viral replication in the B-lymphocyte as (a) both viruses are capable of inducing, with equal efficiency, the formation of preneoplastic lesions containing proliferating B lymphocytes and (b) it appears that equivalent amounts of viral antigen are expressed in the bursae of chicks infected with either virus. We have examined the phenotype of tumors induced by the replication-defective virus REV-T when replicated by the two different helper viruses, REV-A and CSV. In REV-T(REV-A)-infected chicks, the majority of tumors that develop are negative for IgM expression. In contrast, the majority of tumors induced by REV-T(CSV) infection are IgM+. This finding is confirmed by recovery of IgM- cell lines from REV-T(REV-A)-infected chicks and IgM+ cell lines from REV-T(CSV)-infected chicks. In addition, repopulation studies show that bursal-derived cells that are IgM+ serve as target cells for REV-T(CSV)-induced lymphomas. This study demonstrates, therefore, that REV-T can induce IgM+, B cell lymphomas with high efficiency. We conclude that infections by the helper viruses, REV-A and CSV, differ dramatically in their effects on the composition of the population of cells that serve as targets for REV-T-induced neoplasia.

Animals↗
PubMed1988

Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.

Animals↗
PubMed1987

The effect of alterations in myc gene expression on B cell development in the bursa of Fabricius.

Infection of 18-day embryonic bursal lymphocytes with a v-myc-containing retrovirus leads directly to a polyclonal proliferation of surface immunoglobulin-positive (slg+) cells in the bursa of Fabricius detected four weeks after hatching. These v-myc-expressing bursal cells repopulate the follicles of chemically ablated bursae more efficiently than total normal 18-day embryonic bursal cells. In contrast, comparable normal bursal cells lose the ability to repopulate follicles by four weeks. Bursal lymphocytes expressing either a retroviral v-myc or a c-myc gene deregulated by adjacent retroviral integration retain the ability of embryonic bursal lymphocytes to diversify their immunoglobulin light chain genes. These results suggest that retroviral deregulation of myc expression during avian B cell development induces outgrowth of a population of cells with the cardinal phenotypic characteristics of bursal stem cells.

Animals↗
PubMed1986

Selective integration of avian leukosis virus in different hematopoietic tissues.

Hematopoietic tissues obtained from avian leukosis virus (ALV)-infected Hyline SC chickens were analyzed for the presence of integrated viral DNA sequences. Cells were prepared from bone marrow, bursa, spleen, thymus, and peripheral blood. Following the removal of erythrocytes, cellular DNAs from each of these tissues were examined by Southern analysis. During the first few weeks of infection, DNA from the bone marrow contained as many as 0.5 copies of viral DNA per haploid genome. Cells from the bursa and peripheral blood contained between 0.05 and 0.15 copies per haploid genome. In contrast, neither splenic nor thymic DNA contained significant levels of viral DNA sequences even though infected birds developed titers of circulating virus between 10(5) and 10(6) IU/ml of plasma. DNA prepared from erythrocytes isolated from the peripheral blood of these birds contained approximately 0.4 copies of integrated viral sequences per haploid genome at 2 weeks after infection. Despite greater levels of integrated sequences in other tissues, by 9 weeks after infection viral sequences were detected only in DNA from bursal lymphocytes. Cells prepared from the spleen and thymus of infected birds were also examined for their size distribution, their internal complexity and their surface expression of immunoglobulin. None of the populations examined differed from normal, uninfected control preparations. These results suggest that ALV infection occurs primarily in the bone marrow and that the different tissues of the hematopoietic system are selectively infected. Further, these results indicate that ALV infection persists longer in bursal lymphocytes than in other hematopoietic tissues. Previous studies have demonstrated that the lymphoid tumors that develop in white leghorn chickens following ALV infection are bursal-dependent B-cell lymphomas that express immunoglobulin M. The observations presented in this communication offer, in part, an explanation for the restricted phenotype of the lymphoid tumor that develops in the SC chicken. Further, the data suggest an explanation for the bursal-dependent nature of the ALV-induced lymphoma.

Animals↗
PubMed1985

Cell lines derived from avian lymphomas exhibit two distinct phenotypes.

Lymphoid cell lines were derived from three avian leukosis virus (ALV)-induced lymphomas. These cell lines contained proviral DNA sequences integrated upstream from the c-myc proto-oncogene, expressed increased levels of c-myc RNA, and were tumorigenic in syngeneic animals. While cell surface immunoglobulin (IgM) was expressed by all three cell lines, only one of the lines secreted IgM into the culture medium. Further, analysis by light microscopy and flow cytometry demonstrated that these cell lines exhibited two distinct morphological and light-scattering profiles. The two nonsecreting lines exhibited a lymphoblastoid phenotype, whereas, the secreting line possessed a more differentiated plasmacytoid phenotype. These findings implicate the activation of c-myc in the pathogenesis of tumors representing two distinct stages of B-cell differentiation within a single animal species.

Animals↗
PubMed1985

Formation of a transformed follicle is necessary but not sufficient for development of an avian leukosis virus-induced lymphoma.

Avian leukosis virus (ALV) infection of susceptible chickens induces bursal lymphomas after a latent period of several months. The clonal development of these B-cell tumors is believed to be a multistep process. Histopathological changes, referred to as transformed follicles, occur within the target organ soon after virus infection and may represent a proximal stage of lymphomagenesis. To establish further the significance of this lesion and its relationship to the subsequent development of lymphomas, we have compared the incidence of transformed follicles observed in animals susceptible or resistant to ALV-induced tumor development. During the 8 weeks following ALV infection, transformed follicles were detected in 82% of the susceptible animals and in 11% of the resistant animals. These results indicate that the incidence of transformed follicles in these animals correlates with their susceptibility to lymphoma development. Furthermore, each transformed follicle does not develop into a tumor. These observations suggest that the formation of a transformed follicle is necessary but not sufficient for lymphoma development.

Animals↗
PubMed1984

Characterization of endogenous viral loci in five lines of white Leghorn chickens.

Five lines of chickens have been examined for the presence of DNA sequences related to the endogenous avian retrovirus. Five new loci have been identified, based upon analysis with the restriction endonucleases SacI and BamHI. One locus has been associated with the production of infectious endogenous virus. Restriction endonuclease mapping suggested a limited similarity between the flanking cellular sequences of two of these loci, ev-17 and ev-18, and several endogenous loci, including ev-1, already characterized. The data suggested that these two loci might have been generated by chromosomal duplication. Hybridization analysis with a probe containing the cellular sequences that flank ev-1, however, revealed that these flanking sequences shared no detectable homology with the cellular sequences that surround ev-17, ev-18, or nine other endogenous loci that were examined. These results are consistent with the hypothesis that several of the endogenous viral loci resulted either from independent infections of the germ line or from virus transpositions.

Animals↗
PubMed1984

Differential response to avian leukosis virus infection exhibited by two chicken lines.

Infection of susceptible chickens with avian leukosis virus (ALV) results in the development of bursal lymphomas. These neoplasms develop within the bursa of Fabricius following a latent period of several months. The response exhibited by two previously uncharacterized chicken lines to ALV infection has been examined. The two lines, Hyline SC and FP, responded differently to ALV infection. During a 24-week period following intravenous ALV infection, 27 of 50 SC chickens developed bursal lymphomas. No lymphomas developed in the 36 FP chickens tested. A majority of the SC chickens that developed lymphomas also exhibited widespread metastasis to the liver, spleen, and kidneys. Analysis of cellular DNA from the primary and metastatic tumors demonstrated the clonal nature of these neoplasms and revealed altered c-myc loci, as reported in other studies, suggesting the importance of this locus in the development of these tumors. Further characterization of the ALV infection of SC and FP chickens will provide an opportunity to analyze the mechanism of resistance and to contribute to the understanding of the tumorigenic process.

Animals↗
PubMed1984

Replication of endogenous avian retrovirus in permissive and nonpermissive chicken embryo fibroblasts.

Clones of chicken embryo fibroblasts exogenously infected with the endogenous avian retrovirus were analyzed to examine the replication of this virus in permissive (Gr+) and nonpermissive (Gr-) cells. The results demonstrate that the endogenous virus was capable of infecting both Gr+ and Gr- cells with equal efficiency. Infected clones of Gr+ and Gr- cells differed, however, in two significant ways. At the time of their initial characterization, the Gr+ clones produced 100- to 1,000-fold more virus than the Gr- clones. Further, the amount of virus produced by Gr+ clones did not change significantly during serial passage of the cells. In contrast, continued passage of the infected Gr- clones resulted in a gradual increase in the amount of virus produced. Individual clones of infected Gr- cells produced infectious virus at rates that, initially, differed by a factor of more than 10(4). The large differences in the production of virus by these clones could not be explained by equally large differences in the number of infected cells within the clonal populations. Greater than 80% of the clonal populations examined ultimately produced virus at rates that were not significantly different from the rates observed in infected Gr+ cells. Virus produced by these infected Gr- cells exhibited the same restricted replication upon establishing a new infection in nonpermissive cells. Analysis of the appearance of free and integrated viral DNA sequences during endogenous virus infection of Gr+ and Gr- cells demonstrated that, after an initial delay in the synthesis of free viral DNA in Gr- cells, the nonpermissive cells ultimately acquired as many integrated viral DNA sequences as were found in infected Gr+ cells. These results indicate that a majority of the infectious particles of the endogenous virus are capable of establishing infection in a Gr- cell and, ultimately, of producing virus at a rate that is not significantly different from that produced by infected Gr+ cells. The virus produced from the Gr- cells is not a stable genetic variant of the original endogenous virus that is capable of unrestricted replication in nonpermissive cells. The reduced efficiency with which the endogenous virus initially replicates in nonpermissive cells and the increased length of time required for infected Gr- cells to produce maximal virus titers suggest that the endogenous virus may utilize a different mechanism of replication in Gr+ and Gr- fibroblasts.

Alpharetrovirus↗
PubMed1984

Avian leukosis virus infection: analysis of viremia and DNA integration in susceptible and resistant chicken lines.

Avian leukosis viruses induce lymphoid leukosis, a lymphoma which develops within the bursa of Fabricius several months after virus infection. Chickens from the Hyline SC and FP lines are, respectively, susceptible and resistant to avian leukosis virus-induced lymphoid leukosis. We examined plasma and cellular DNA obtained from avian leukosis virus-infected chickens for the presence of viremia and integrated viral sequences to determine whether the extent of virus infection is comparable in individuals of both lines. A less than twofold difference in the frequency of viremia was detected between chickens of the two different lines. Although the analysis of plasma samples, which were obtained at different times postinfection, demonstrated that the duration of viremia was comparable in both susceptible and resistant chickens, the onset of the viremia observed in susceptible chickens generally preceded by 1 week that observed in resistant chickens. Moreover, integrated viral sequences were detected in approximately 90% of the SC and 40% of the FP chickens. The appearance of infectious virus in the plasma was, in general, associated with the presence of integrated viral sequences in both the bursal cells and the erythrocytes obtained from the same chicken. The presence of both the viremia and the integrated viral DNA sequences was transient, suggesting a mechanism for the elimination of virus-infected cells in both susceptible and resistant chickens. Furthermore, at 5 weeks postinfection no integrated exogenous viral sequences were detected in splenic lymphocytes obtained from either chicken line, regardless of whether these chickens were viremic or had integrated viral sequences detectable in other tissues. Our results indicate that extensive avian leukosis virus replication occurs in approximately 50% of the FP and 100% of the SC chickens. Although it appears that the viral infection spreads more quickly in the SC chickens, our results afford no obvious explanation of the resistance to the development of lymphoma exhibited by FP chickens.

Animals↗
PubMed1981

Rous sarcoma virus infection of synchronized cells establishes provirus integration during S-phase DNA synthesis prior to cellular division.

Synchronized chicken embryo fibroblasts, prepared by addition of serum to stationary cells arrested in Go, were exposed to the Prague strain of Rous sarcoma virus. At different times during the cell cycle, high molecular weight DNA was prepared from infected cells and examined for the presence of newly integrated viral DNA sequences. The results demonstrate that newly integrated viral sequences were first detected during S-phase DNA synthesis 9 hr after infection. The presence of colchicine prevented cellular division and delayed the appearance of progeny virus but it did not affect the appearance of viral specific DNA in the high molecular weight fraction of cellular DNA. Our results indicate that provirus integration, occurring during S-phase DNA synthesis, does not require cell division. Previous experiments have demonstrated that Rous sarcoma virus infection of chicken embryo fibroblasts requires cell division to initiate viral RNA synthesis and the production of progeny virus. The findings presented in this report support the hypothesis that division of the infected cells is required for an event that controls viral expression at the level of the integrated provirus.

Animals↗
PubMed1981

Independent expression of avian sarcoma virus in doubly infected chicken embryo fibroblasts.

Infection of a chicken cell with avian sarcoma virus requires division of the infected cell before synthesis of infectious progeny is initiated. This requirement for a cell division for the complete expression of avian sarcoma virus has been examined further with chicken embryo fibroblasts infected with two distinct viruses. Chicken cells infected with and producing a mutant of Rous sarcoma virus temperature sensitive for transformation (tsLA24PR-A) were arrested in G0 by depletion of serum factors from growth medium. These stationary cells continued to produce infectious progeny in the absence of further cell division. Superinfection of the stationary cells with the wild-type Prague strain of Rous sarcoma virus (PR-RSV-C) produced a stable double infection in these cells. Progeny of the superinfecting PR-RSV-C, however, were not detected until these cells underwent division after stimulation with fresh serum-containing medium. The addition of colchicine to these serum-stimulated cells, although not affecting production of the tsLA24PR-A, inhibited the appearance of progeny of the superinfecting PR-RSV-C. These experiments indicate that each avian sarcoma virus infection of a chicken embryo fibroblast requires division of the infected cell for production of that virus regardless of whether or not the cell is already producing a similar virus. The results suggest, therefore, that the requirement for a cell division represents a requirement for an event that controls virus expression in a "cis-acting" fashion specific for the provirus.

Animals↗
PubMed1981

Clonal analysis of the integration and expression of endogenous avian retroviral DNA acquired by exogenous viral infection.

Rous-associated virus-0 is one of several endogenous avian retroviruses that are transmitted vertically and that can be isolated from different inbred lines of chickens. These viruses, referred to here as induced-leukosis viruses bearing a subgroup E glycoprotein (ILV-E), are all closely related. Clonal populations of fibroblasts from line 15B and line 100 inbred chickens have been examined for the presence and expression of exogenously acquired ILV-E sequences. Restriction enzyme analysis of uniform populations of line 15B fibroblasts, prepared by cloning cells either before or after infection with ILV-E, indicates that viral sequences were inserted at multiple sites within the cell genome. Analysis of 49 clonal populations of line 100 fibroblasts containing between one and five copies of exogenous ILV-E sequences demonstrated that each clone was characterized by a unique set of viral DNA insertions within the cell genome. The expression of the exogenous ILV-E sequences within these fibroblast clones was examined by using reverse transcriptase activity as a measure of virus production. Some clones produced an amount of virus equivalent to that produced by an equal number of the uncloned ILV-E-infected parental fibroblasts. Other clones produced 5- to 10-fold less virus. Still other clones produced no detectable virus at all. Among nine clones derived from cells containing a single copy of the ILV-E provirus, the level of virus production differed more than 100-fold. DNA from these clones was analyzed with several different restriction endonucleases to characterize the location and arrangement of the ILV-E sequences. All nine clones consisted of cells that appeared to contain a complete provirus inserted (i) in a different site within the cellular DNA and (ii) in an orientation that was colinear with the viral genomic RNA. It was observed that several cleavage sites potentially affected by methylation were equally available for cleavage in all clones regardless of the level of viral production.

Animals↗